I! l W WI! I"! I w «l: UNI Jill“ I b I ll! LWLIZ’ATION OF SH-fiukiifilfikflimh‘. EWC‘JSPHA‘I‘E 591’ RUMWANTS AS B'ETERI'NNE BY METABOMSM HUME i’hasia kw Ma Degree: oi: M. S. MICHEGAH S‘FA‘E'EE UNIVERSITY Donald Kayser 1960 Ti "":“ Lfil‘.~H-’ M LIBRARY Michigan Sues University UTILIZATION OF DI-AMMONIUM PHOSPHATfl BY RUMINANTS AS DETdRMINED BY NMHnBOLJSM STUDIES By Donald Keyser A THSSIS Submitted to the College of Agriculture of Michigan State University of Agriculture and Agplied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIfiNCE Department of Dairy ' 1960 / ' - . 4 ‘4” ‘ / ‘ //é//<;V/7%71?ffiexi sifCI Approved QM ‘n w '..;7 —253 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. C. A. Lassiter, Dairy Department, for his guidance and assistance throughout this work and for the critical reading of this manuscript. I would also like to thank Dr. R. S. Emery, Dairy Department, for his guidance in the laboratory work and for his critical read- ing of this manuscript. TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . .-. 3 Urea Utilization by Ruminents . . . . . . . . . . . . . . . . . 3 Phosphorus Metabolism in Cattle . . . . . . . . . . . . . . . . 9 DESIGN OF EXPERIMENT . . . . . . . . . . . . . . . . . . . . . . . . 13 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 19 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 REFERLNCthjO LIST OF TABLES TABLE Page I. WEIGHT GAINED AND BLOOD UREA NITROGEN LEVELS OF CALVES . . . . 5 II. CALCULATED VALUES OF THE RATIONS FED . . . . . . . . . . . . . lh III. RECORD OF WHEN THE cows HERE DUE AND FRESHENING DATE . . . . . 16 Iv. EFFECT OF THE VARIOUS RATIONS ON THE NITROGEN BALANCE AND DIGESTIBILITY (C/CON DAILY) . . . . . . . . . . . . . . . . 2O v. EFFECT OF THE RATIONS ON BLOOD UREA NITROGEN VALUES . . . . . 23 VI. BLOOD SERUM INORGANIC PHOSPHORUS VALUES FOR THE VARIOUS RATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . 25 VII. THE EFFECTS OF THE RATIONS ON PHOSPHORUS RETENTION AND BALANCE . . . . . . . . . . . . . . . . . . . . . . . . . . 26 INTRODUCTION Several years ago, it was demonstrated that ruminants could convert non-protein nitrOgen into protein and incorporate it into their bodies. Since that time, many products have been evaluated to estab- lish their value as a substitute for natural proteins. Nest of the products evaluated were ammoniated compounds such as urea, biuret, ammonium bicarbonate, and dicyandiamide. It has been demonstrated that these products have a protein sparing effect that varies con- siderably with the compound tested. Di-ammonium phosphate is similar to the compounds mentioned above and ruminants should be able to utilize the nitrOgen of this product. The nitrOgen content of di-ammonium phosphate is about 21 per cent and has approximately one half the nitrogen content of urea. In addition, di-ammonium phosphate contains 30 per cent phosphorus. Since the phosphorus is in the form of a soluble phosphate, it should prove to be a very effective phosphorus supplement. The manufacturers believe that di-ammonium phosphate may have a place in the commercial feed industry if it can be used both as a source of phosphorus and nitrogen for ruminants. Preliminary feeding trials by Lassiter (17) showed that di- ammonium phosphate was palatable when fed to dairy cattle at the levels of l, 2, and h per cent of the grain mixture. Similar palata- bility trials by Shaw (29) indicated that di-ammonium phoSphate was palata- ble up to 3 percent of the grain mixture. Mono-ammonium phosphate was not as palatable in this study and was not recommended for cattle. Both authors indicated that the palatability of the grain mixtures de- creased as the level of di-ammonium phosphate increased. There was no Sign of toxicity from this product in either trial. The next phase of this study was to conduct a digestion and nitrogen balance trial to compare the utilization of the nitrogen in di-ammonium phosphate with the nitrogen of urea and soybean oil meal. Phosphorus balance and blood inorganic phosphorus levels were used to indicate the fate of the phosphorus in this product. REVIEW OF LITERATURE There appears to have been little previous work with di- ammonium phosphate as a feed for ruminants. Since there is insu- fficient information on this product, it is the desire of the author to review the literature on the utilization of urea as a non-protein nitrogen source for animals, and the phosphorus metabolism of cattle. It is the author's belief that a review of literature in these two areas will provide the information needed to evaluate the results accumulated in the metabolism trial, where di-ammonium phosphate was compared with urea and soybean oil meal. Urea Utilization by Ruminants The research on urea has been quite extensive andis used by many as a standard when comparing other non-protein nitrogen products as a source of nitrogen for animals. Work has been completed with both non—ruminant and ruminant animals. Nest of the work has been with ruminants where the utilization of these products shows promise of having some economic value. In addition, "in vitro” studies have been completed to reveal more information On how these products are utilized by the animals. There is little evidence that urea will ever be of much value to non—ruminants. Work with rats by Rose and co-workers (26) has shown that urea and other ammoniated products can be utilized to some extent when only the essential amino acids were fed to them but they could not utilize enough urea for normal growth. Liu and co-workers (19) studied the metabolism of urea in pigs with Labeled (N15) nitrogen in low protein ration. A small but definite amount of this urea was incorporated into the body. Hays et a1. (12) fed different levels of urea to swine. Slightly higher daily gains were recorded with 0.16 per cent and 0.31 per cent urea in the rations. These gains were small and less efficient. They required more grain per pound of gain, with the feed efficiency decreasing as the urea content increased. When the urea content reached the equivalent of 10 per cent of the protein, it reduced daily gains. These and other studies indicated that this type of feed is of little value to single stomach animals. Hart et a1. (9) compared the utilization of urea and ammonium bicarbonate to casein in growing calves. The calves received milk for the first two weeks and then.were gradually changed over to a complete dry ration. The basal ration was approximately a 6 per cent protein ration and was supplemented with the different sources of nitrogen to raise the nitrogen content of the ration to the equivalent of an 18 per cent protein ration. All the calves receiving the extra nitrogen gained more than the calves on the basal ration, indicating that some of the nitrogen from the non-protein sources was being utilized. The calves on the casein gained substantially more than the other groups. The results of the trial are summarized in TABLE I. Loosli and McCay (21) studied the utilization of urea by two month old calves. The calves were fed milk for a month and gradually changed over to dry feed. The basal ration was a h.h per cent protein ration. This was increased to a 16.2 per cent protein ration with the 5 addition of urea or natural proteins. Calves fed on the normal ration gained 80 pounds, while the calves on urea rations gained 6l pounds. This trial indicated that urea was utilized by the calves but not as efficiently as natural protein. TABLE I WEIGHT GAINED AND BLOOD UREA NITROGEN LEVEI.OF CALVES Type of Protein Pounds Of Gain Mg._% of Blood Urea Nitrogen 28 weeks E0 weeks Before exp. End of exp. Basal Ration 120 201 16.67 7.2 Ammonium bicarbonate 180 276 , . lh.2O 17.2 Urea 220 290 15.58 19.7 Casein 312 M27 lh.2o 17.2 Bartlet and Cotton (2) studied the effect of urea as a substitute for part of the protein for young cattle from 7 to 17 months of age. When 0.127 pounds of urea was added to the rations, it accounted for a 0.2h pound increase in their daily gains indicating that the cattle were utilizing urea. In this experiment, natural proteins produced better gains but they were not significantly greater than those on urea rations. The blood urea nitrOgen levels for all animals were fairly close except those on low protein diets. The blood urea nitrOgen levels were lower on these animals. USing young heifers, Parham et al. (2%) compared urea, ammoni- ated molasses, and cotton seed meal as a source of nitrogen. These heifers were from 19 to 33 months of age. Urea and ammoniated molasses 6 made up a third of the protein in an 18 per cent crude protein ration. The average daily gains in pounds were: urea 0.9h, ammoniated molasses 1.0hl, and cotton seed meal 1.23. The results were not significantly different because of the wide variations in gains within groups. Davis et al. (5) used four groups of milking cows to compare a low protein ration, soybean oil meal, ureaiand di-cyandiamide as a protein source in two 56 day trials. The non—protein nitrogen re- placed one third of the protein in the urea and dicyandiamide rations. The low protein ration had two-thirds the nitrogen Value of the other rations. There was no significant differences in body weight changes, milk production, or blood urea nitrOgen levels. Rust et al. (27) studied the utilization of urea and dicyandiamide as a nitrogen source for low protein rations with 2% cows over a 305 day period. Approximately one-third of the nitrogen.was of a non- proteinsource. Average milk production for these groups were: soy— bean oil meal 2h.h, urea 22.3, and dicyandiamide 22.2 pounds. The average weight gained or lost by these groups were 43, -28, and -86 pounds respectively. The differences in the milk production was not significant but the cows fed urea and soybean oil meal lost signifi- cantly less body weight than the cows fed dicyandiamide. The average blood urea nitrogen levels of this group were: soybean oil meal 6.3, urea 6.88, and dicyandiamide h.07 mg. per cent. This experiment indicated that urea was utilized fairly well but not dicyandiamide. The blood urea nitrogen levels indicated that the dicyandiamide had a low ammonia activity in the rumen. Archibald et al. (I) studied urea as a partial substitute for natural proteins. TWenty-eight Holstein cows were used in a three year year trial to determine the adequacy of urea. The grain ration con- tained 3 per cent urea. The urea furnished #2 per cent of the nitrogen in the grain and 25 per cent of the total nitrogen in the ration. Milk yields and weight changes were not significantly different but milk yields and weight gains were generally a little better when natural proteins were fed. Blood urea nitrogen levels were slightly higher when the cows were fed urea. High level urea feeding by Lassiter and co-workers (18) indi- cated that milk yields and body weight gains would decline slightly as urea supplied 0, 30, 50, and 70 per cent of the total nitrogen in the grain ration. These differences were not statistically significant. Protein digestability values for the rations were: soybean oil meal 53.5, 30 per cent urea nitrogen 50.1, 50 per cent urea nitrogen 50.h, and 70 per cent urea nitrogen 51.8 per cent. The blood urea nitrogen values for the different rations were: 8.8, 10.7, lO.h and 11.2 mg. per cent respectively. Ward et a1. (30) and Thompson et al. (28) also fed urea and soybean oil meal to cattle with about the same results. The natural, proteins seemed to be a little better but the differences were not large enough to be statistically important. Wagner et a1. (31) used a fistulated heifer to study the effects of urea on the ingesta of the rumen. Protein levels averaged l to 2 per cent higher in the ingesta when urea was fed in addition to a basal ration of 8.h5 per cent protein. The urea was hydrolized in one hour and the ammonia disappeared in h—6 hours. In another experiment Wegner et al. (32) compared the utilization of urea at different protein levels. When the grain mixture was over 18 per cent, the efficiency of urea CD utilization decreased. In this study urea was utilized fairLy efficiently up to 12 per cent protein equivalent with an 11.5 per cent basal ration composed of natural proteins. The conversion of urea decreased when the protein of the ingesta of the rumen became greater than 12 per cent. In the digestion of nutrients, Belasco (3) indicated that urea increased cellulose digestion. This work indicated that the microflora that digest cellulose need a readily available supply of ammonia for Optimum activity. "In vitro“ studies by Wegner et a1. (33) showed that microflora can convert inorganic nitrogen to protein. “They found that Optimum pH for the microflora to be 5.5 to 7. The carbohydrates used in the medium were corn molasses, cerelose (commercial glucose), starch, and cellulose. All the sources of carbohydrates were readily utilized by the bacteria that converted ammonia to protein except cellulose. The reSponse with cellulose was very poor. In this study, ammonium bicarbonate was just as effectively utilized as urea. Pearson and Smith (25) indicated that the ability of the ruminant to convert urea to ammonia was very high. One hundred grams of rumen ingesta could convert 100 grams of urea to ammonia in one hour. During the incubation of urea and ingesta with an available starch supply, it was possible for the mdcroorganisms to fix 0.8 mg. of nitrogen per 100 gm. of rumen liquor in 2h hours. At this rate it would be possible for the intact animal to fix 72 grams of nitrogen or #50 grams of protein. Starch proved to be a better source of carbohydrate than glucose, galactose, sucrose, or other sugars. 9 Studie U} by Bell et al. (A) indicated that the source of starch did not affect the utilization of urea when it was from corn, de- hydrated sweet potatoes, barley, milo, cane molasses, or corn and molasses. The cane molasses and urea combination had the poorest nitrOgen retention while corn was the highest. The digestability of barley increased a little when soybean oil meal was fed with barley instead of urea. Phosphorus Metabolism in Cattle Phosphorus metabolism is quite difficult to determine in cattle. Most of the phOSphorus is excreted in the feces and is mixed with the undigested ph sphorus. Urine Phosphorus is usually quite low, amounting to less than 0.1 of a gram per day at times (8). When determining the requirement of phosphorus, it has been the practice to determine the phOSphorus balance. The different sources of phosphorus were fed and the requirement determined by how much was needed to keep a constant phosphorus equilibrium. Another factor studied was the inorganic blood phosphorus levels. This has been shown to vary some with the food supply and there is some evidence that it varies with the supply of phosphorus (23). Studies on the effects of normal physio- logical functions such as eating, exercise, and rest were completed to determine their effect on the blood inorganic phosphorus level. Many investigators have studied the requirement of phosphorus in cattle. Lamb et a1. (1.6) fed high and low phOSphorus rations to rattle. Rations supplemented with bone meal enabled some heavy milk producers to have a positive balance. The cows on low phosphorus rations lost their appetite, and in general were in a negative phosphorus lo balance. The range of phosphorus retained or lost Varied from +C.lh to +lC.l3 grams per day for the high phosphorus rations and from —11.85 to +8.70 grams for the cows on the low phosphorus rations. The cattle on the high phosghorus rations had a blood inorganic phosphorus level ranging from 5.3 to 7.AA mg. per cent while cows on the low phosyhorus diets were below A mg. per cent Most of the phosphorus was excreted in the feces. Urine excretion averaged about 0.17 grams per day with a range of 0.07 to 0.5A grams. Huffman and co—workers (1h) studied the phosphorus requirement of cattle when alfalfa hay was the principle source of protein. Alfalfa hay is low in phosphorus and has an adverse ratio between calcium and phosphorus. These studies indicated that the absorption of phosphorus was better if the ratio of calcium to phosphorus was 1.25:1. On low phosphorus rations, blood levels of inorganic phosphorus were 1.85 to 2.3 mg. per cent in calves and as low as 1 mg. per cent in mature cattle. When phOSphorus was decreased in the rations, the blood level of phosphorus dropped from around 8 mg. per cent to less than 5 mg. per cent in four days. When the phosphorus supplement was withdrawn from lactating cows to study their phosphorus reserve, the blood level drOpped from about 5 mg. per cent to 3.51 mg. per cent in one week, to 1.95 mg. per cent the second week, and to 1.16 mg. per cent the third week. After several months when milk production drOpped, the blood level of phosyhorus increased to over 5 mg. per cent. Work reviewed by Forbs (8) indicates that the cattle varied in their absorption and retention of phosphorus. In one study the phos- phorus pentoxide (P205) retention varied from -A0.0 to *hl.8 grams. ll Hart et a1. (1L) fed high and low amounts of tran ash to milkiné cows. The high ration contained 130 grams of P205 and the low ration ess than 50 grams of P205. The balance of phosphorus ranged from ~13 grams to +12% grams of P205. Sekles et al. (7) studied phosphorus deficiencies in cattle. ‘. . fl ,— Elood phosphorus levels on low phosphorus rations were 0.77 to 3. CI" mg. per cent for inorganic phosphorus with a balance ranging from +0.05 to —h.03 grams of phosphorus. The high phosphorus rations had a phosphorus balance ranging from $2 to +lh.h1 grams per day. When cattle on low phosphorus rations were given a phosphorus supplement, the blood levels of inorganic phOSphorus raised from 1 to 2.5 mg. per cent to about 6 mg. per cent in five days. Blood phosphorus levels varied from 2.8M to 8.13 ms. per cent during this five day period. Work by loosli et a1. (20) indicated that phosphorus require— ments for cattle varied with age and milk production. Results indi- cated that heifers needed about 1.8 grams of phosphorus per 100 pounds of body weight for the first year, 1.7 grams per 100 pounds the second year and 1.3 grams per 100 pounds for the third year. Growing calves retained from 3.6 to 5.3 grams of phoSphorus per day. Cows require about 1 gra per 100 pounds of body weight for body maintenance and from 0.5 to 0.7 grams per pound of milk. Kleiber et al. (15) measured endo;enous phosphorus in cattle with P32. The work indicated that endogenous phosphorus varies with the supply of phosphorus and other factors. In this trial, the endogenous phosphorus from the feces varied from #3 to 70 per cent of the total phosphorus excreted. The amount excreted depended to some extent on the supply of phosphorus in the feed. Palmer et a1. (23) worked on blood phosphorus levels of cattle as influenced by food intake, exercise, and time of day. Blood inor— ganic phosphorus levels before feeding ranged from 5.83 to 8.13 mg. per cent. Little change was recorded after eating but the blood inorganic phosphorus level did increase slightly in about one hour and decreased to normal in about three hours. This increase was about a 0.h mg. per cent increase. Exercise increased the phosphorus level for a short time followed by a decrease for a short period. This work indicated that the time to draw blood for phosphorus determinations was after the cattle had rested for several hours and before they had been fed. Paturition causes a rapid drOp in blood phosphorus level, and the calf will have a higher blood phosphorus level than the dam. Young calves have a higher blood phosphorus level than mature cattle and is usually around 8 mg. per cent. Mature cattle usually average about 6 mg. per cent with a range of h to 8 mg. per cent for inorganic blood phosphorus. DESIGN OF EXPERIMENT This experiment was designed as 3 8h day metabolism study to compare the utilization of the nitrogen in di-ammonium phosphate to that of soybean oil meal and urea, and to investigate the value of the phosphorous in this product. Four cows were fed each ration in the same sequence as they appear in TABLE II. The tentative feeding schedule was a 17 day feeding period for each.ration. This was divided into;a 10 day adjustment period and a 7 day collection period. Fecal and urine samples were taken daily during the metabolism trial and total.weights recorded. Blood samples were taken at the end of the adjustment period and the end of the collection period. The experiment was started February 7, 1959 and completed June h, 1959. The tentative date for completion was May 1, but was not possible because the cattle had a tendency to go off feed when di-ammonium phosphate was introduced or increased in their grain mixtures. Three factors may have been involved in the cows refusal of their grain mixture that contained di—ammonium phosphate. Di-ammonium phosphate did not seem to be as palatable as urea and the cattle never consumed their grain mixture as readily when this product was in their grain. 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It is evident that if the two urine deter- minations were lower, as one would expect from the other urine deter- minations, the nitrOgen retention for the two rations would have been fairly close. The digestibility of the di—ammonium phosphate rations drOpped about 10 per cent below the urea and soybean oil meal rations. This drOp is quite large and indicates that di-ammonium phosphate may not be utilized as readily as urea. The digestibility of the protein in the different rations were: basal ration 32.h per cent, soybean oil meal 55.2 per cent, urea 57.0 per cent, urea and di—ammonium phosphate #7.0 per cent and di-ammonium phosphate hh.7 per cent. The basal ration had the lowest digestibility of the five rations. The addition of di-ammonium phosphate increased the digestibility coefficient but was not equal to the soybean oil meal and urea rations. The large increase in the fecal nitrogen was not expected. Itvould be reasonable to expect that a soluble product like this to be absorbed in the digestive tract and excreted in the urine if it was not utilized. Instead, there was a large increase in fecal nitrOgen indicatiqgthat some of this product may be passing through the digestive tract without being absorbed. There was no evidence as indicated by the blood urea nitrogen levels, that the ammonia concentration or activity in the rumen was especially low with the exception of the basal ration. The blood urea nitrOgen level for the basal ration was very low. There was no evidence in the literature that blood urea nitrOgen levels were ever recorded as low as the ones in this experiment. TABLE V summarizes the results of the Jill Lr. 31%|! R) {‘0 blood values for urea nitrogen. There Was some doubt that the results of the urea test were accurate as there were very large readings from the blanks. The readings varied from day to day and ranged from about lO to 50 per cent of the blood urea determinations. The level of blood urea nitrogen before the experiment was at its highest peak and was never as high again except for about three of the blood tests taken while urea was being fed. In the literature large variations in the blood urea levels have been noted. Archibald (1) recorded a slightly higher levels of blood urea while feeding urea. For urea, it averaged about ll mg. per cent while regular feeds were about 10.27 mg. per cent. Rust et al. (27) had lower values in his trials with soybean oil meal 6.3, urea 6.8 and dicyandiamide h.07 mg. per cent. Davis et al. (5) had blood levels as follows: Test I - low protein 12.3, soybean oil meal 13.5, urea lh.O, dicyandiamide l2. TEst II - low protein 8.1, soybean oil meal 8.l, urea 10.0, and di- cyandiamide 8.1 mg. per cent. Archibald (1) indicated that normal values of urea nitrOgen were lb to 23 mg. per cent. Most of the literature indicated that the blood level of urea nitrOgen was 6 to l2 mg. per cent with older cattle and 15 to 20 mg. per cent for calves. In this trial, there was enough phosphorus in every ration to meet the requirements of the cows. Di-calcium phosphate Was used as the phosphorus supplement. The phosphorus supplement was not eliminated in the last two rations even though the di-ammonium phOSphate supplied more phosphorus than the cows needed. It was thought that if every ration had the same source of phOSphorus with the exception of the di-ammonium phosphate rations any change in blood inorganic phOSphorus or phosphorus retention could be attributed to the di-ammonium phosphate. The main fault of this experiment is that itvas not designed to compare the availability 23 psomohmoh muoneso woo opp “defined psoEpmSnom map no one one pm vamp dooap one poomonmon mumpess oo>o one .UOHpom eoHpooHHoo one mo one one pm pmop dooap one a ms.m so.m so.e me.m oe.m me.m mm.o mwom oa.m mH.m we.m ommno>< mm.m mm.m mm.m sw.m mamua ow.m ms.s ms.s mm.m soa-< wo.m mm.m mm.m ss.s mm.m mm.m mm-s sate mm.m mm.m mm.m om.> sm.m sm.: sm.m Hm.s mm.m ms.a 0:.s mam mm.m os.s sm.: om.m om-HH mm.m Hm.m oa.m mm.m me.a sa.oa oaa-e ms.s mm.m we.» sm.m mm.m H>.m mm.oa mm-< & .m: .oz 300 AH OH m m s w m s m m a Hecapmafishopon nose mood manage Satan womb , Eomm Hmmmm whomom cofipmm eaten may: mMDA¢> zmoomBHz ¢mmb QOQHm zo MZOHB¢m mmB mo BUMhmm > mammfi of the phosphate is the di—ammonium phosphate with other phosphorus supplements. The blood serum levels of the inorganic phosphorus increased substantially in this trial when di-ammonium phosphate was fed. The blood phosphorus level was nearly equal for the first three rations and the initial blood test. This was in the h to 8 mg. per cent range which is considered normal for cattle. When the di—ammonium phosphate r was fed, the blood inorganic phosphorus level increased 80 to 100 per cent. This is fairly conclusive evidence that the phosphorus is readily available to the cattle and was being absorbed. A complete summary of the blood serum inorganic phosphorus determinations are listed in TABLE VI. Very little can be determined in this trial about how the phosphorus was absorbed or utilized. In all rations, there was slight positive phosphorus retention except the last ration in this test. In the last ration, where the phosphorus level fed was about three times that recommended by Morrison's standards for dry cows, (22), there was a fairly high retnetion of phosphorus. This averaged nearly 10 grams a day for the cows while the other rations had a positive balance ranging from +1 to +h grams of phosphorus. TABLE VII summarizes the phosphorus balance for this study. One strange development in this study was the high phosphorus excretion in the urine of A—107 while on the di-ammonium phOSphate ration. This cow excreted over 8 grams of phosphorus per day which is abnormal for cattle. In all the determinations, there was a fairly high phosphorus concentration in the urine compared to the levels found by other investigators. 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